SUMMARY Current stroke research focuses more on understanding the brain?s self-protective and repair mechanisms. Detailed elucidation of these mechanisms is crucial as such knowledge could lead to development of therapeutic interventions which mimic or engage the brain?s self-protective/repair mechanisms and can lead to successful stroke therapy. With the proposed research we seek to develop potent and selective ?drug-like? small molecule activators of peptidase neurolysin (Nln) which will be used as research tools and lead chemical entities to move the drug discovery process forward for development of a novel class of drugs. Recently published and pilot studies from our laboratory have identified Nln as one of the brain?s self- protective mechanisms, functioning towards preservation and recovery of the brain after stroke. Functional significance of Nln in the post-stroke brain is based on its ability to inactivate several neurotoxic and generate three cerebro- protective/regenerative neuropeptides, which are known from numerous experimental and clinical studies to critically contribute to the outcome of stroke. Based on this evidence we view Nln as a central peptidase involved in brain restorative mechanisms following stroke. In this collaborative application we will leverage our expertise in multiple aspects of the drug discovery process and will develop potent ?drug-like? small molecules which can selectively enhance the catalytic efficiency of Nln and can be used as experimental therapeutic agents for post-stroke brain protection and recovery. This proposal has been formulated based on our compelling experimental data indicating that catalytic activity of Nln can be enhanced by two structurally related dipeptides and a distinct non-peptide chemotype. Feasibility of the proposed studies is shown by our initial structure-activity relationship studies of the identified Nln activators and by in vivo experiments in two different mouse stroke models indicating that inhibition of endogenous Nln after stroke aggravates stroke injury, whereas overexpression of Nln in the brain or its delivery to the post-stroke brain substantially improves stroke outcome. The goals of this proposal will be accomplished in three well-integrated aims: (1) design and characterize a diverse and proprietary library of compounds based on three active hits and guided bioassays to identify critical functional residue interactions within the Nln binding site, and to develop high-potency, brain-permeable, selective activators of Nln with ?drug-like? properties; (2) conduct biochemical and structural studies to characterize the activation mechanism that the identified Nln activators exploit; (3) determine the therapeutic potential of Nln activators in post-stroke brain protection and recovery using a mouse model of stroke. This work is highly innovative because there are no activators of Nln described in the scientific literature and such compounds were never considered to have therapeutic potential. The collaborative investigative team, comprising experts in medicinal chemistry and drug discovery, crystallography and structural biology, enzyme biochemistry and pharmacology, blood-brain barrier physiology and stroke pharmacology, is highly qualified to conduct the proposed studies. Our long-term goal is to translate the lead Nln activators from bench to bedside and develop an effective therapy, which would transform the current treatment modalities for a vast number of stroke patients.